Heavy Metal Tolerance In Fungus

The heavy metal pollution in the soil is one of the major threats to the environment due to its accumulative nature and non- biodegradability. Both natural such as geologic parent material, volcanic eruptions, wind - blown dusts in deserts and anthropogenic such as agricultural, mining, sewage water, industrial, transportation sources can be the reason of heavy metal pollution in environment (Vaclavikova et al., 2008;. Luo et al., 1997). From total contents of heavy metals in the soil, 95% of As has been reported to came from Wastewater irrigation whereas the large fractions of Ni (60%), Mn (88%), Cr (75%), V (76%) were originated from the natural sources and the major proportions of Cu (81%), Zn (70%) and Pb (93%) were reported to be accumulated from industrial origins (Dong, B et al., 2019). The metals or metalloids having specific gravity larger than 5 g cm-3 and possessing atomic number greater than 20 can be considered as heavy metals (Rascio and Navari-Izzo 2011). The metals such as Cu, Mn, Fe, Ni, Zn are known to be essential metals as they are required for the different biological functions (cofactor for enzymes, stabilizing cell structure) which can be toxic for the cells above their threshold limits (Bruins et al., 2000). On the other hand, metals such as Cd, Pb,Cr, Hg are considered to be non-essential as they are not required for cell metabolism and are highly toxic for cells even in trace amounts (Haferburg and Kothe 2007). Starting from soil to plants the metal can enter into higher tropical levels of food chains such as insects, herbivores, humans which may results in the imbalance of ecosystem and food chain. The recent work showed the Pb transfer from eggplant and Tomato plants- mealybug- Cryptolaemus montrouzieri (beetle) food chains. The Pb content transferred in Tomato is higher as compared to eggplant and reduction in Pb concentrations was observed at successive tropic levels illustrating Pb bio-minimization (Zang et al., 2017). The continuous intake of metal polluted crops may cause severe issues in humans such as inhibition of ATP production, protein coagulation by Arsenic; kidney, liver, brain malfunctioning by lead, capillary damage, corrosion, by copper and bloody urine, diarrhoea, vomiting by Zinc. The ground water is also at risk of heavy metal contamination by the leaching of metals from top layers of the soil which can be further transferred into potable water (Shi et al., 2019).

For the remediation of heavy metal pollution, different chemical techniques such as reverse osmosis, precipitation, oxidation, reduction, ion- exchange have been used to treat the heavy metal pollution which is costly, produce unsuitable by products in the environment and mostly applicable to small areas only (Macauley and Hong 1995; and Vasarevicius, 2005). On the other hand biological remediation is considered to be state-of-the-art technique which can be used to reduce the metal pollution from soil ecosystem without causing any harmful effects to the environment in cost effective manner. Heavy metal detoxification has been reported by micro- organisms such as bacteria [such as Bacillus thuringiensis (biosorption), Pseudomonas sp. (metal accumulation)], fungus (such as Pisolithus albus, Suillus species) or algae (such as Chlorella vulgaris, Pseudochlorococcum typicum, Phormidium ambiguum (Cyanobacterium) (Piotrowska-Niczyporuk et al., 2012; Shanab et al., 2012; Oves et al., 2013; Ahemad and Malik 2011; Colpaert et al., 2011; Reddy et al., 2016). Among different micro- organisms mycorrhizal fungi are the class of fungi which can establish the symbiotic association with plant roots. About 70% of the flowering plants around the globe are associated with Arbuscular mycorrhizal fungi (AMF) and 3% plants, chiefly woody plants, are associated with ectomycorrhizal fungi (ECM) (Smith and Read 1997; Smith & Read, 2008). The ultramafic soil gives us the number of evidences showing the role of mycorrhizal associations in the evolutionary adaptations of plant against extreme heavy metal stress conditions in soil. In ultramafic soils of Neo Caledonia, the dominant symbiosis of ECM with plants showed to be the major reason of their enhanced tolerance against extreme soil conditions (Prin et al., 2012). The inoculation of Pisolithus albus, an ECM fungus, isolated from the ultramafic soils of New Caledonia with Eucalyptus globulus and Acacia spirorbis resulted in the increased transfer of deficient essential elements from soil to host plants and their protection from harmful high concentrations of heavy metals. These experiments have also reported that some isolates of Pisolithus albus inoculated with host plants are more efficient in increasing plant biomass as compared to others. It has also been reported that ultramafic soils do not minimize but promotes the ECM diversity (Branco and Ree, 2010; Moser et al., 2009).

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The ECM fungi have developed different mechanisms to detoxify the high concentrations of heavy metals in the soil. The release of ECM exudates containing different chelating agents, heavy metal sequestration in vacuoles, cell wall barriers and transcriptional regulation of genes are some of the mechanisms used by ECM against heavy metal stress (Shi et al., 2019). Many genes associated with metal stress tolerance in mycorrhizal fungi have been characterised in past few years. By functional yeast complementation assays, two membrane transporter encoding genes OmZnT1 and OmFET have been identified from mycorhizzal fungus Oidiodendron maius, which are responsible for restoring Zn tolerance in Zn sensitive mutant strains of Saccharomyces cerevisiae. The homology based BLAST searches revealed their relation with cation diffusion facilitators and iron permease subfamilies respectively. The movement of Zn ions in endoplasmic reticulum by OmZnT1 and increasing magnesium ions influx into cell by OmFET to counteract Zn toxicity were the suggested mechanisms responsible for Zn tolerance (Khouja et al., 2013).At gene level, both arbuscular and mycorrhizal fungi have tendency to regulate the gene expression, transport, absorption, sequestration and detoxification of Heavy metal related genes. In ECM fungus Suillus luteus, the transcript level of SIZRT1which has role in uptake of Zn, was found to vary with the different concentrations of Zn in medium. SIZRT1 rRNA level raised in the absence of Zn in the medium whereas the level of mRNA get reduce with the increased concentrations of Zn (Coninx et al., 2017). The SIMTa and SIMTb metal ions chelating genes of Suillus letus got transcribed in higher amounts under the elevated concentrations of Cu2⁺ ions (Nguyen et al., 2017). There are many reports giving evidences for the significant role of metallothionein proteins in metal homeostasis. Metallothionein (MT) protein was first discovered by Margoshes and Vallee in 1957 from horse kidney cortex and has cadmium binding efficiency which results in the accumulation of cadmium in the tissue (Margoshes et al., 1957). MTs are the family of low molecular weight Cysteine- rich conserved proteins, which which binds to metal ions through thiol group of their cysteine residues, playing prime role in ion homeostasis (Cobbett and Goldsbrough 2002). In order to find the mechanism behind the potential of accumulating Zn in sporocarp by ECM fungus Russula atropurpurea, size-exclusion chromatography (SEC separation) has been performed with extract collected from sporocarps. It was observed that approximately 80 % of extracted Zn was found to be in complex with 5 kDa cysteine rich peptides identified as RaZBP1 and RaZBP2 genes homologous to metallothioneins were responsible for providing Cd and Zn tolerance to yeast hypersensitive mutant strains (Leonhardt et al., 2014).In ECM fungus Amanita strobiliformis, AsMT1 (having three different 1a, 1b, 1c isogenes), AsMT2, AsMT3 have been characterised as metallothioneins by BLAST searches of sporocarp transcriptome with known metallothioneins of different micro- organisms. The techniques QT-PCR and yeast complementation assays revealed the induced gene expression of former genes in the presence of Cu/Ag, Cd and Zn/Cd respectively (Hložková et al., 2016). Similarly In Laccaria bicolour, the transcript level of metallothioneins LbMT1 and LbMT2 was found to be induced in the presence of Cd and Cu respectively (Reddy et al., 2014).

Another widely studied bio molecule is glutathione which is chief component of fungal defence system against metal stress. Glutathione is tripeptide molecule, L-γ-Glutamyl-L-Cysteinyl-Glycine, found in all mammalian tissues possessing anti-oxidating, thiol group maintenance and detoxifying activities (Lu, 2009). Glutathione are well known for their potential to prevent oxidative stress and xenobiotics detoxification in cells in which Glutathione-S-transferase catalyses the binding of Xenobiotics to glutathione to form conjugates which can be actively secreted out from the cell through ATP-ase pump (Sheehan et al., 2001). In order to explore impact of cadmium stress on γ-glutamylcysteine synthetase gene expression in ECM fungus Hebeloma cylindrosporum, the real time PCR analysis had been carried out. The positive correlation was observed showing induction of the higher Hcγ-GCS gene expression over increased concentration of Cd, which ultimately results in larger glutathione biosynthesis (Khullar and Reddy 2019). With phytochelatin synthase as catalyst, glutathione can also act as the precursor for the phytochelatin synthesis which is the part of fungal defence system against heavy metal stress conditions (Hematy et al., 2019).

The extensive studies have been carried out to characterized mycorrhizal fungal genes associated with metal tolerance but complete gene expression profiles of ECM fungi under metal stress have not been attempted yet which makes this plot valuable to explore more. In past years the microarray hybridization technique was used widely to identify differentially regulated genes in an organism under some specific conditions. By this methodology, the attempts were made to identify the differentially expressed, Cd stress related genes in ECM Paxillus involutus based upon the hybridisation of labelled cDNA microarrays of control and P. involutus under Cd stress conditions on nylon membrane. The differentially expressed genes identified and sequenced followed by their homology searches which were further quantified by the QT- PCR with gene specific primers. The results showed the induced expression of laccase, polyphenols, glutamine encoding gene (helps in thiols synthesis such as gluthathione), aconitase (enzyme related to tricarboxylic acid cycle) and depressed expression of threonine dehydratase and hydrophobins in P. involutus exposed to Cd. The upregulation of metallothionein was related to increased complexation of Cd with metallothionein whereas the dowregulation of hydrophobins were related to diversion of cysteine for Cys rich components (Jcob et al., 2004). The hybridisation-based microarray techniques requires prior sequence knowledge and can also create false positive results with unspecific hybridization (Casneuf et al. 2007). The technique RNA- Seq has been evolved to overcome the limitations of previously used gene expression quantification techniques such as hybridization based microarray techniques, Sanger sequencing of expressed sequence tag libraries, cap analysis gene expression (CAGE) and so on (Kukurba et al., 2015).

In present time, instead of being using microarray hybridization and other technologies, more advance and high throughput technique named as RNA- Seq. RNA- Seq technique is used which is revolutionized by the development of next generation sequencing technologies and is successful in monitoring the gene expression levels, allelic specific expression and alternate splicing (Wang et al., 2009; Kukurba et al., 2015).

This technology was used to identify the effect of different lignocelluloses compositions of different trees on total gene expression of soft wood degrading fungus Phanerochaete carnosa. The total RNA was isolated from each P. carnosa culture growing in symbiosis with different wood substrates and digested with DNase for quality improvement followed by cDNA synthesis. The FAST- Q files containing sequenced cDNA reads were generated by Illumina next generation sequencing. 63 % of total 152 million paired end reads were mapped to 10,257 gene models of P. carnosa by Maker genome annotation and Augustus gene prediction tools out of which 88% of 10, 257 were allocated with gene functions by homology based searches. Further map2slim.pl tool was used for gene automated annotations and for exploring evolution of DNA sequenes phylogenetic analysis was done. The most abundantly expressed genes with predicted functions such as cellulose binding protein, lignin peroxidise, chitin synthase, xylanases and so on were subjected to qRT-PCR technology for further verification where GO Slim analysis was performed for categorising differentially expressed genes into specific categoris (MacDonald et al., 2011). Similar methodology had been used to understand the plant biomass digesting mechanism of anaerobic fungi Orpinomyces sp. strain residing in alimentary canal of herbivores. RNA- Seq analysis of fungus cultured on four different lignocellulosic biomasses identified expressed enzymes and their transcription levels on each biomass which further helps in revealing the constituent and overlapping functions of some enzymes (Couger et al., 2015).

Hence from above explained data we have collected enough evidences about the great potential of ECM fungus Pisolithus albus to tolerate metal stress conditions. Till date, many genes responsible for proving tolerance to ECM fungus P. albus have been characterised but complete profiles of genome wide gene expression of fungus under metal stress has not been reported yet and from data showed above, RNA- Seq is concluded to be the most advance and efficient technology for genome wide transcriptomics analysis. This makes the complete transcriptomics analysis of ECM fungus P. albus under metal stress with RNA- Seq appropriate and valuable approach for further research.